1. Field of the Invention
This invention relates to a method for measuring distributions and changes of concentrations of ions, in a cell, which contribute to controlling of functions, etc. of the cell.
2. Related Background Art
In quantifying concentrations of free ions, such as calcium ions, etc., which are present in a live cell, fluorescence probe dyes, such as Fura-2, Indo-1, etc., are used. The methods of synthesis and characteristics of these dyes are described in "The Journal of Biological Chemistry, Vol. 260, No. 6, pp. 3340-3450 (1985)" and "Biophysical Journal, Vol. 54, pp. 1089-1104 (1988)". These probe dyes have a characteristic that they bond with and dissociate from certain ions to have variable fluorescence characteristics. Due to this characteristic, a probe dye is applied into a cell, and an intensity of fluorescence generated by an excitation beam is measured, whereby an ion concentration in the cell can be measured.
The measurement based on excitation beams at only one wavelength and fluorescence of only one wavelength cannot give accurate measured values when distribution of a probe dye in the cell is not uniform. In addition there is a possibility that changes of a fluorescence intensity, such as attenuation of fluorescence, etc., which are independent of an ion concentration are measured together.
To make up for this disadvantage a method has been employed in which a fluorescence probe dye having an isosbestic point is used to measure either fluorescence intensities corresponding to excitation beams at two different wavelengths, or intensities of fluorescence at two wavelengths generated by an excitation beam of one wavelength, and the ratio of the fluorescence at the two wavelengths is determined.
FIGS. 1A and 1B show examples of the prior art, demonstrating excitation spectra and emission spectra of a fluorescence probe dye used in the above-described method. FIG. 1A shows excitation spectra of Fura-2 for measuring a concentration of calcium ions in samples, and the figures above the curves depicting the spectra indicate calcium ion concentrations in samples. As shown, a characteristic of Fura-2 is that it bonds with calcium ions in a cell to increase the fluorescence intensity in response to an excitation beam of 340 nm wavelength. Conversely, its fluorescence intensity is decreased by an excitation beam of 380 nm wavelength. Another characteristic of Fura-2 is that an intensity of the fluorescence generated by an excitation beam of 360 nm wavelength is independent of concentrations of calcium ions. In the case where Fura-2 is used, a calcium ion concentration is given based on a fluorescence intensity ratio between, e.g., 340 nm and 380 nm, or 340 nm and 360 nm, and on a calibration curve prepared beforehand. The calibration curve for converting a value of the fluorescence intensity ratio into an absolute value of a concentration can be obtained by preparing a dye containing calcium ions of a known concentration, introducing a fluorescence probe dye thereinto and measuring a fluorescence intensity ratio.
FIG. 1B is a fluorescence spectra of Indo-1 and shows spectra at respective wavelengths of fluorescence generated when a cell is irradiated with an excitation beam of 355 nm wavelength. Indo-1 has an isosbestic point at around a fluorescence wavelength of 440 nm, as does Fura-2. Accordingly, in the same way as the above-described case using Fura-2, a calcium ion concentration in the cell can be given based on a fluorescence intensity ratio between two wavelengths of the fluorescence generated by the irradiation of an excitation beam of one wavelength.
Measuring devices for use in the above-described measuring methods using fluorescence probe dyes are spectrofluorometers, microphotometers combining fluorescence microscopes and photomultipliers, image analyzers combining fluorescence microscopes and television cameras, etc.
The above-described methods are elaborated in "A. Miyakawa et al., Bunseki Kagaku, Vol. 38, No. 11, pp. 842-649 (1989)", and "A. Miyakawa, Photomedical and Photobiology, Vol. 13, pp. 15-18 (1981)".
In the above-described dual wavelength fluorescence measuring method, an error deriving from a non-homogenous concentration of a probe dye introduced into a cell can be corrected by calculating a fluorescence intensity ratio.
The above-described method is usable in cases where a probe dye is reactive only with certain ions, but it is considered that in a cell the probe dye bonds also with protein, membrane components, etc. which are present there in high concentrations. Accordingly fluorescence spectra of the probe dye, and a chelating constant thereof with ions adversely change. Then a problem with the dual wavelength fluorescence measuring method is that an error resulting from such interactions between the dye and components other than ions cannot be corrected, and an accurate ion concentration cannot be given.